The focus of this proposal is to understand how a pluripotent cell commits to a specific fate at the earliest stages of differentiation. Cell identity is determined by the transcriptional potential of its genomic DNA. This is achieved, in large part, through specific chemical modifications of DNA and chromatin that epigenetically programs gene activity. A critical aspect of epigenetic programming is DNA methylation, which "hard-wires" the genome by limiting transcriptional output to those programs that control cellular specialization. Because this occurs most actively in stem cells, the mouse ES/EB system is especially valuable for capturing the complete and ordered programming of a pluripotent genome in its early stages and in quantities that are amenable to analyses. We propose to purify a discrete population of hematovascular progenitor cells from differentiating mouse ES cells and compare the DNA methylation and RNA expression profiles with those of differentiated progenitors that do not possess a hematovascular potential. In this way, we can discriminate between DNA methylation patterns that are associated with differentiation in general and those that are specific to the hematopoietic lineage. We will use a blended high-throughput epigenetic/genic/bioinformatic platform to identify the initial sites of DNA methylation within a genome as it transitions from pluripotency to a committed state, couple this information with RNA expression analyses, and compare it with similar data obtained from cells of distinct developmental potential. Functional verification of hematovascular-specific methylated DNA regions that we identify will be performed in ES cell differentiation assays to examine the biological role of these regions in cell lineage commitment. By employing genomic tools, we hope to establish a basis for how cells codify their cell fate choices. An important aspect of our work is its possible application to cell replacement therapy for blood diseases. For example, long-term replacement of the hematopoietic system will necessitate transplantation of the most immature cells, such as those derived from hematovascular progenitor cells. Our work will generate biochemical quantities of highly purified populations of this important precursor, which will be used to define its epigenomic signature through DNA methylation and RNA expression profiling. We hope to apply this platform to unambiguously identify the precise epigenetic characteristics of any cell within a population that is to be evaluated for its therapeutic potential.